Unfolding and Aggregation of mAbs - …

1 1 Unfolding and Aggregation of mAbs Application Note NT-PR-005 Analysis of formulation-dependent colloidal and conformational stability of monoclonal antibodies Franziska S ltl1, Jonathan Derix1, Patrick Garidel2, Michaela Blech2 and Dennis Breitsprecher1 1 NanoTemper Technologies GmbH, Munich, Germany 2 Boehringer Ingelheim Pharma GmbH & Co. KG, Global Bioprocess & Pharmaceutical Development, Global Formulation Development, Biberach, Germany Abstract The growing number of biological drugs such as monoclonal antibodies (mAbs), as well as the wealth of heterogeneity between mAb variants requires a thorough development process to maximize mAbs compliance with regulation.

2 Therefore, biophysical analytical methods are required already at early stages of the development process to guide and streamline further antibody processing and to predict antibody developability. In this case study, we demonstrate how the Prometheus can be used to predict long-term mAb stability in a formulation screen by simultaneous quantification of both, conformational and colloidal stability of biologicals in thermal gradients. Introduction Monoclonal antibodies (mAbs) constitute the majority of therapeutic biologicals today.

3 Owed to their high specificity and potency, they are used to treat a number of diseases, ranging from different cancer types to autoimmune defects. Novel protein engineering approaches lead to a growing number of therapeutic mAbs, which can additionally be modified to be bispecific, conjugated with other biologicals or modified with small molecule drugs. The conformational and colloidal stability of antibodies are key parameters to predict their stability and also developability, since they affect the long-term storage stability which is critical for drug approval [1, 2].

4 The growing number of mAbs and mAb variants in development pipelines calls for Figure 1: Relation between conformational and colloidal stability of antibodies. (A) Equilibrium and non-equilibrium states between folded and unfolded antibodies and aggregates. (B) Schematic plot showing the consequence of strong Aggregation of the unfolded state. Irreversible Aggregation leads to an accumulation of aggregates over time, eventually resulting in complete Aggregation of the mAb.

5 (C) Schematic representation of directed Unfolding and Aggregation of antibodies in a thermal gradient. 2 biophysical methods which can quickly assess those parameters [3, 4]. Screenings for conditions and antibody constructs in early stages of development aim to identify most promising candidates to meet regulatory requirements for drug approval. Here, we use a small scale formulation screen for a therapeutic monoclonal IgG1 antibody to demonstrate the capability of the Prometheus in determining critical stability parameters.

6 One approach to determine the conformational stability of proteins is to follow their Unfolding in temperature gradients [5-9]. Increasing the temperature of the sample results in a transition of proteins from the folded to the unfolded state (Figure 1C). The temperature at which this transition occurs, the Tm, is used as a surrogate parameter for the thermal stability of the protein. The Prometheus uses nanoDSF to assess mAb conformational stability in thermal gradients by following changes in tryptophan fluorescence emission [8, 9].

7 Owed to the high sensitivity of the fluorescence detection in capillaries, even minute changes in antibody conformation can be detected, allowing for the unambiguous identification of multiple Unfolding events which can be attributed to the different mAb subdomains. At the same time, the Prometheus can detect changes in colloidal stability and temperature-induced Aggregation . This is achieved by detection of the backreflection intensity of a light beam that passes the sample twice (Figure 2).

8 Figure 2: Schematic representation of the backreflection principle to detect protein Aggregation . (Left) light passes the capillary, is backreflected into the detector and light intensity is quantified. (Right) particles scatter light, leading to an extinction of the incident light and thus a reduction of backreflected light, which is a direct measure for protein Aggregation . Upon Aggregation , the intensity of the backreflected light decreases due to light scattering, and thus serves as a measure for total Aggregation in a sample.

9 Importantly, backreflection and fluorescence are detected simultaneously, allowing for maximal scanning speed and data point density. A simultaneous assessment of conformational and colloidal stability is a powerful approach to predict long term stability. Every antibody irrespective of its conformational stability is in equilibrium between native (folded) and unfolded conformational states (Figure 1A). If the unfolded state of the antibody shows a strong tendency to form aggregates, it precipitates out of solution, resulting in more antibody becoming unfolded driven by the law of mass action (Figure 1B).

10 Thus, strong Aggregation of unfolded antibodies has to be prevented, by identifying optimal formulation compositions or molecular modifications that prevent Aggregation . Simultaneous backreflection and fluorescence analysis using the Prometheus provides several important information: Firstly, it is possible to directly correlate thermal and colloidal stability, meaning that Aggregation -causing Unfolding events can be identified, and more importantly, Aggregation onset temperatures can be determined.